Einarsson Elin, Troell Karin, Hoeppner Marc P, Grabherr Manfred, Ribacke Ulf, Svärd Staffan G
Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden.
Department of Microbiology, National Veterinary Institute, Uppsala, Sweden.
PLoS Negl Trop Dis. 2016 Mar 25;10(3):e0004571. doi: 10.1371/journal.pntd.0004571. eCollection 2016 Mar.
Differentiation into infectious cysts through the process of encystation is crucial for transmission and survival of the intestinal protozoan parasite Giardia intestinalis. Hitherto the majority of studies have focused on the early events, leaving late encystation poorly defined. In order to further study encystation, focusing on the later events, we developed a new encystation protocol that generates a higher yield of mature cysts compared to standard methods. Transcriptome changes during the entire differentiation from trophozoites to cysts were thereafter studied using RNA sequencing (RNA-seq). A high level of periodicity was observed for up- and down-regulated genes, both at the level of the entire transcriptome and putative regulators. This suggests the trajectory of differentiation to be coordinated through developmentally linked gene regulatory activities. Our study identifies a core of 13 genes that are consistently up-regulated during initial encystation. Of these, two constitute previously uncharacterized proteins that we were able to localize to a new type of encystation-specific vesicles. Interestingly, the largest transcriptional changes were seen in the late phase of encystation with the majority of the highly up-regulated genes encoding hypothetical proteins. Several of these were epitope-tagged and localized to further characterize these previously unknown genetic components of encystation and possibly excystation. Finally, we also detected a switch of variant specific surface proteins (VSPs) in the late phase of encystation. This occurred at the same time as nuclear division and DNA replication, suggesting a potential link between the processes.
通过包囊化过程分化为感染性包囊对于肠道原生动物寄生虫贾第虫的传播和生存至关重要。迄今为止，大多数研究都集中在早期事件上，而晚期包囊化的定义尚不明确。为了进一步研究包囊化，聚焦于晚期事件，我们开发了一种新的包囊化方案，与标准方法相比，该方案能产生更高产量的成熟包囊。此后，我们使用RNA测序（RNA-seq）研究了从滋养体到包囊的整个分化过程中的转录组变化。在整个转录组和假定的调控因子水平上，上调和下调的基因都观察到了高度的周期性。这表明分化轨迹是通过发育相关的基因调控活动来协调的。我们的研究确定了13个基因的核心，这些基因在初始包囊化过程中持续上调。其中，有两个基因构成了以前未表征的蛋白质，我们能够将其定位到一种新型包囊化特异性囊泡上。有趣的是，在包囊化后期观察到最大的转录变化，大多数高度上调的基因编码假定的蛋白质。其中几个基因被表位标记并定位，以进一步表征这些以前未知的包囊化（可能还有脱囊化）遗传成分。最后，我们还在包囊化后期检测到变异特异性表面蛋白（VSP）的转换。这与核分裂和DNA复制同时发生，表明这些过程之间可能存在联系。
